Microarray technology has led to significant advances in many areas of medical and biological research,[1] opening up avenues for the combinatorial screening and identification of single nucleotide polymorphisms (SNPs),[2] high sensitivity expression profiling of proteins,[3,4] and high throughput analysis of protein function.[5] However, current microarraying technologies, such as spotting with pin arrays, inkjet printing or methods derived from photolithography, are limited in their practical resolution—from hundreds to tens of microns, depending on the technique. The density of the fabricated arrays and therefore the number of distinct deposited biological entities (which include but are not limited to proteins, nucleic acids, carbohydrates, lipids, and especially complex or assemblies thereof, such as viruses or cell components) are therefore limited. There is therefore a need for a method capable of few-microns and sub-micron-scale arraying of said biological entities.
In addition, the dimensions of each site in currently fabricated microarrays are typically much larger than the size of individual biological molecules or assemblies being deposited. Therefore, large numbers of said entities are present in each site and only the statistical, collective behavior of these ensembles can be studied. Isolated biological particles have been deposited at random on solid substrates by e.g. contacting a very dilute solution of said biological particles with a solid substrate with carefully selected surface chemistry during a carefully selected amount of time, optionally followed by rinsing steps. This method offers little if any control over the density and placement of said particles. There is therefore a need for a miniaturization method—down to the nanometer length scale—with the potential to “site-isolate” nano- and microscale biological entities at the single-particle level with precise positioning. With such method, new opportunities will be available to the biochemical and biomedical research communities to begin to study such entities individually rather than collectively. Site-isolation is of commercial interest, e.g. in pharmaceutical R&D during drug discovery—especially to rapidly elucidate the fundamental mechanism of interaction between a drug candidate and its target—without need for difficult and time-consuming techniques like crystallization and X-ray analysis. Other commercially relevant single particle biology experiments include, for example, studying (a) the effect of the relative orientation between biological entities on their interactions; (b) the cooperative behavior of a selected number of pathogens infecting simultaneously e.g. a cell; (c) the binding of a single antibody with an antigen or conversely the cooperative behavior of multiple antibodies towards an antigen; (d) variations in the interaction of a target drug with individual forms of a polymorphic protein or that of various members of the same protein family; and (e) the effect of genetic variations among viruses in an array on their interaction with another biological item.
Prior to this invention, advances have been made in immobilizing virus particles on templates created by DPN and micro-contact printing.[11,12] However, a need exists to provide the ability to chemically control the position of the immobilized virus structures at the single-particle level. This is in part because of limited resolution (vide supra), the size of the particles interrogated, and especially the chemistry used to immobilize them. Indeed, prior efforts have focused on the genetic modification of a virus particle to present unnatural surface binding functionality to the patterned interface.[11,12] For reasons of cost and scalability, it is preferable to avoid manipulation of said viruses. There is therefore a need for a method to chemically control the position of the immobilized virus structures at the single-particle level without need for chemical or genetic modification of said viruses.
No admission is made that any of the references cited in this Background section or thereafter are prior art.